The long-term goal of this proposal is to identify the mechanism of human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC) nuclear localization in infected cells. Previous work identified amino acid residues in the viral matrix, integrase and Vpr proteins that functioned in nuclear localization specifically in nondividing cells, and more recent findings identified novel nuclear localization signals in the central DNA flap made by reverse transcription and integrase residues Val-165 and Arg-166 that functioned in both dividing and nondividing cells. However the Preliminary Studies described in this proposal refute the roles of these novel sequences in nuclear translocation. Viruses carrying mutations in the central DNA flap replicated under a variety of conditions. Although defective flap function was identified in primary T-cells, there was no evidence for a nuclear import defect. Although integrase mutants V165A and R166A were acutely defective, these viruses were primarily integrase-defective, not import defective. It was determined that the integrase mutants fell into a category of previously-described pleiotropically defective mutants, leading to the hypothesis that defective nuclear import is a phenotype common to a variety of defective integrase mutants, and that these mutants are primarily defective for intracellular trafficking as compared to nuclear membrane translocation. Thus, pleiotropic import-defective integrase mutants will be used to determine intracellular steps essential for HIV-1 replication complexes to reach the chromosomal targets of integration. Residues involved in specific nuclear import of HIV-1 PICs will be identified following extensive mutagenesis screens. Host cell factors essential for intracellular trafficking to chromosomes and PIC translocation through intact membranes will be identified using protein-protein interaction assays. The results will determine host protein-HIV-1 interactions essential for PIC nuclear import and intracellular trafficking, which should define targets for the development of novel antiviral drugs against essential steps in the HIV-1 life cycle.